Fragile X syndrome (FXS) occurs through mutation of the FMR1 gene and is a common form of heritable intellectual disability. The FMR1 protein (FMRP) modulates neuronal function via binding and regulation of select RNA molecules. Recent studies show that FMRP function is more complex and includes roles in mediating a replication stress-induced DNA damage response (DDR) via direct binding to chromatin. The interaction of FMRP with chromatin comes from its ability to bind a subset of methylated histones via the function of its tandem Agenet domain, but more detailed mechanisms by which FMRP interacts with chromatin to modulate the DDR, whether there are additional functions for chromatin-associated FMRP, and the impact these properties have on behavior phenotypes of FXS are very much unknown. To conduct the proposed studies, we use the fruit fly D. melanogaster as a model, exploiting the ability to visualize chromatin at high resolution in the form of polytene chromosomes, the amenability of the organism to molecular genetic manipulations, and the fact that fruit flies have an ortholog of FMR1 (dfmr1; dFMRP) which shares all known functional domains with its mammalian counterparts. Moreover, flies mutant for dfmr1 exhibit behavior phenotypes with significant parallels to those observed in FXS patients. In the first Aim of this proposal, we will explore a connection between chromatin insulators and dFMRP. Recent work from my laboratory has expanded insulator function to include modulating changes in chromatin structure necessary for DNA replication and repair, and in response to exogenous stresses. These results suggest a functional link between chromatin insulators and dFMRP that may further explain how FMRP exerts an effect on chromatin. We have performed experiments to test whether dfmr1 and the insulator protein-encoding gene suppressor of Hairy wing [su(Hw)] function in common pathways, and find that a loss-of-function mutation of dfmr1 can suppress the wing margin development phenotype of ct6, a mutation mediated by the binding of Su(Hw) in the insulator sequences of a gypsy transposon inserted between the promoter and wing margin enhancer of the cut (ct) gene. This result is supported by finding that dFMRP and Su(Hw) protein co-localize on polytene chromosomes. We will characterize this interaction to discern the mechanism(s) by which dFMRP suppresses the ct6 phenotype, using existing alleles of dfmr1 that selectively disrupt chromatin or RNA binding domains of dFMRP. We expect that these studies will illuminate novel mechanisms by which dFMRP regulates chromatin function. In the second Aim, we address a connection between the chromatin-binding Agenet domain of dFMRP and behavior phenotypes of FXS, using techniques for controlling temporal and spatial expression of dfmr1, and the robust memory and circadian locomotion phenotypes of dfmr1 mutants that have parallels with the behavior deficits of FXS patients. The studies in this proposal may give insights into mechanisms of FMRP nuclear function that are necessary to develop additional therapeutic avenues for FXS.